Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-1 (ET-1), an endothelial cell-associated paracrine factor. These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers. It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells. ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the timing and location of Purkinje fiber differentiation within the embryonic myocardium.     

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.     

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.     

Evidence for altered ETB receptor characteristics during development and progression of ventricular cardiomyocyte hypertrophy

The hypothesis that endothelin (ET) receptor mechanisms are altered during development and progression of left ventricular hypertrophy (LVH) in vivo was tested using spontaneously hypertensive rats (SHRs). Ventricular cardiomyocytes were isolated from SHRs before onset (8 and 12 wk) and during progression (16, 20, and 24 wk) of LVH and compared with age-matched normotensive Wistar-Kyoto (WKY) rats. PreproET-1 mRNA expression was elevated in SHR (P < 0.05) relative to WKY cardiomyocytes at 20-24 wk. ET binding-site density was twofold greater in SHR than WKY cells at 12 wk (P < 0.05) but normalized at 20 wk. ET(B) receptors were detected on SHR cardiomyocytes as early as 8 wk and their affinity increased progressively with age (P < 0.05), whereas ET(B) receptors were not detected on WKY cells until 20 wk. ET-1 stimulated protein synthesis with similar maximum responses between strains (21-30%), in contrast with sarafotoxin 6c, which stimulated protein synthesis in SHR (13-20%) but not WKY cells at 12-20 wk. In SHR but not WKY cells, the ET(B) receptor-selective ligand A-192621 increased protein synthesis progressively with the development of LVH (15% maximum effect). In conclusion, the presence of ET(B) receptors (8-12 wk) coupled with functional responsiveness of SHR cells but not WKY cells to sarafotoxin 6c at 12 wk supports the involvement of ET(B) receptors before the onset of cardiomyocyte hypertrophy, whereas altered ET(B) receptor characteristics during active hypertrophy (16-24 wk) indicate that ET(B) receptor mechanisms may also contribute to disease progression.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno-and enzyme histochemical study

Summary The differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in bovine hearts was studied with specific antibodies against M-line proteins (MM-creatine kinase and myomesin) and with enzyme histochemistry (succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase). MM-creatine kinase was detected at an earlier stage in Purkinje fibres and atrial myocytes than in ordinary ventricular myocytes. The findings are in agreement with previous ultrastructural observations that an earlier appearance of a dense M-band occurs in Purkinje fibres than in ordinary ventricular myocytes. Myomesin was detected in all three cell types even at early foetal stages, in accordance with suggestions that it is an integral component of the myofibrillar structure. The activity of succinate dehydrogenase gradually increased in both ordinary ventricular and atrial myocytes, while the activity of mitochondrial glycerol-3-phosphate dehydrogenase was high at different stages of early foetal development in the two tissues, finally becoming low in the adult stage. The activity of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase seemed to remain unchanged in the Purkinje fibres from early to late foetal stages. The present study shows that the Purkinje fibres are already different from ordinary ventricular myocytes at early foetal stages and that the two cell types differentiate in different ways. It is concluded that there are also developmental differences between ordinary ventricular and atrial myocytes.     

Cell-specific properties of type V and type IX adenylyl cyclase isozymes in 293T cells and embryonic chick ventricular myocytes

The cDNAs for types V and IX adenylyl cyclases were cloned from a chicken heart library and expressed in 293T cells (plasmid transfection) and in embryonic chick ventricular myocytes (adenovirus infection). Expression of type V or IX cyclases in 293T cells resulted in increases in basal and isoproterenol (ISO)-stimulated cAMP levels, whereas the expression of type V, but not type IX, cyclase increased forskolin (FK)-stimulated cAMP levels. Expression of type V cyclase in cardiac myocytes increased basal and FK-stimulated cAMP levels, variably increased ISO-stimulated cAMP levels, and decreased the content of beta-adrenergic receptors (betaARs). The expression of type IX cyclase in cardiac myocytes increased basal and ISO-elevated cAMP levels and, surprisingly, increased the cAMP-elevating effect of FK. The finding that FK responses are increased in cardiac myocytes but not in 293T cells expressing the type IX cyclase suggests that the host cell influences the properties of the type IX isozyme.     

Inositol 1,4,5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers capable of releasing Ca2+ from sarcoplasmic reticulum/ER subcompartments. The mRNA encoding the intracellular IP3 receptor (Ca2+ channel) has been detected in low amounts in the heart of various species by Northern blot analysis. The myocardium, however, is a heterogeneous tissue composed of working myocytes and conduction system cells, i.e., myocytes specialized for the beat generation and stimulus propagation. In the present study, the cellular distribution of the heart IP3 receptor has been investigated. [3H]IP3 binding experiments, Western blot analysis and immunofluorescence, with anti-peptide antibodies specific for the IP3 receptor, indicated that the majority of Purkinje myocytes (the ventricular conduction system) express much higher IP3 receptor levels than atrial and ventricular myocardium. Heterogeneous distribution of IP3 receptor immunoreactivity was detected both at the cellular and subcellular levels. In situ hybridization to a riboprobe generated from the brain type 1 IP3 receptor cDNA, showed increased accumulation of IP3 receptor mRNA in the heart conduction system. Evidence for IP3-sensitive Ca2+ stores in Purkinje myocytes was obtained by double immunolabeling experiments for IP3 receptor and cardiac calsequestrin, the sarcoplasmic reticulum intralumenal calcium binding protein. The present findings provide a molecular basis for the hypothesis that Ca2+ release from IP3-sensitive Ca2+ stores evoked by alpha 1-adrenergic stimulation is responsible for the increase in automaticity of Purkinje myocytes (del Balzo, U., M. R. Rosen, G. Malfatto, L. M. Kaplan, and S. F. Steinberg. 1990. Circ. Res. 67:1535-1551), and open new perspectives in the hormonal modulation of chronotropism, and generation of arrhythmias.     

Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isl1 in chick embryo

Cardiomyocytes are generated from the precardiac mesoderm and the size of the heart increases dramatically during embryogenesis. However, it is unclear how differentiation and proliferation correlate in the cardiac cell line during development. Here, we show that cardiomyocytes re-entered into a proliferative state after differentiation with a concomitant cell cycle arrest in chick embryo. The cells in the course of differentiation from Isl1-positive cardiac precursors to cardiomyocytes did not proliferate, but differentiated cardiomyocytes proliferated even after the acquisition of contractile function. After differentiation, cardiomyocytes developed a proliferative potential to contribute to the increase in cell numbers during heart development. Almost all differentiated cardiomyocytes (82.8%) incorporated bromodeoxyuridine (BrdU) in vitro, indicating the ability of DNA replication. Furthermore, mitotic chromosomes were observed in the cardiomyocytes in which a sarcomeric structure was sustained in the cytoplasm. We conclude that the sequential events of the differentiation to contractile myocytes and the re-entry into the cell cycle are strictly regulated during cardiac cell maturation. These results provide an insight into the maturation mechanism of the cardiac cell line.     

Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum (SR) during excitation–contraction (e–c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18°C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca²⁺-influx, rather than CICR, during e–c coupling.     

Phosphoinositide 3-kinasegamma (PI3Kgamma) controls L-type calcium current (ICa,L) through its positive modulation of type-3 phosphodiesterase (PDE3)

The modulation of L-type calcium current (ICa,L) is mainly due to mediators acting through activation of G protein-coupled receptors (GPCR) and different protein kinases; among them, phosphoinositide 3-kinasegamma (PI3Kgamma) has been recently discovered to play an important role in the regulation of cardiac contractility and beta-adrenergic signal transduction. Recent reports have demonstrated that, in the heart, different subtypes of beta-adrenergic receptors are coupled to both Gi and/or Gs proteins. While beta1-adrenergic receptors (beta1-AR) couple only to Gs and evoke a strong ICa,L, beta2-adrenergic receptors (beta2-AR) can activate both Gs and Gi proteins and trigger only a limited ICa,L. Here we demonstrate that (i) PI3Kgamma-/- ventricular myocytes are characterized by an higher basal ICa,L density, even if the responsiveness of adenylyl cyclase to Forskolin is comparable to that observed in PI3Kgamma+/+ cardiomyocytes; (ii) both in basal conditions and after beta-AR stimulation, the activity of phosphodiesterase (PDE) type 3 depends on PI3Kgamma; (iii) in PI3Kgamma-/- cardiac myocytes, specific stimulation of beta2-AR is followed by a increase in ICa,L stronger than in wild-type controls. Taken together, our results suggest that the higher values of ICa,L observed both in basal conditions and after beta-AR stimulation in PI3Kgamma-/- ventricular myocytes are mainly due to a positive modulation of PDE3 activity exerted by PI3Kgamma. As observed in PI3Kgamma-/- neonatal cardiomyocytes, cells lacking PI3Kgamma are more sensitive to stimulation of beta2-adrenergic receptors.     

Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.     

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.     

Increased endothelin-1 and endothelin receptor expression in myocytes of ischemic and reperfused rat hearts and ventricular myocytes exposed to ischemic conditions and its inhibition by nitric oxide generation

Endothelin-1 (ET-1) and nitric oxide (NO) exert opposite effects in the cardiovascular system, and there is evidence that the NO counters the potential deleterious effects of ET-1. We investigated whether NO affects the increased mRNA expression of ET-1 and endothelin receptors induced by (i) 30 min of ischemia with or without 30 min reperfusion in myocytes from isolated rat hearts or (ii) ischemic conditions (acidosis or hypoxia) in cultured rat neonatal ventricular myocytes. Ischemia with or without reperfusion produced more than a twofold increase in mRNA expression of ET-1 as well as the ET(A) and ET(B) receptor (P < 0.05), although these effects were completely blocked by the NO donor 3-morpholinosydnonimine (SIN-1; 1 microM). To assess the possible factors regulating ET expression, myocytes were exposed to acidosis (pH 6.8-6.2) or to hypoxic conditions in an anaerobic chamber for 24 h in the presence or absence of SIN-1. At all acidic pHs, ET-1 and ET(A) receptor mRNA expression was significantly (P < 0.05) elevated approximately threefold, although the magnitude of elevation was independent of the degree of acidosis. These effects were completely prevented by SIN-1. ET(B) receptor expression was unaffected by acidosis. Hypoxia increased ET-1 as well as ET(A) and ET(B) receptor expression threefold (P < 0.05), although this was unaffected by SIN-1. Our results demonstrate that myocardial ischemia and reperfusion upregulate the ET system, which is inhibited by NO. Although increased expression of the ET system can be mimicked by both acidosis and hypoxia, only the effects of the former are NO sensitive. NO may serve an endogenous inhibitory factor which regulates the expression of the ET system under pathological conditions.     

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.